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Environmental fate & pathways

Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental starting date: 14-02-2017 Experimental completion date: 16-03-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Batch/Lot number: 0708501022
Purity: 99.8%
Appearance: red solid particles
Solubility in water: insoluble in water
Stability: not relevant
Storage: at ambient temperature in the dark
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Secondary activated sludge (09-02-2017) was obtained from the wastewater treatment plant Nieuwgraaf in Duiven, The Netherlands. This plant is an activated sludge plant treating predominantly domestic wastewater. The activated sludge was preconditioned to reduce the endogenous respiration rates. To this end, 0.40 g Dry Weight (DW)/L of activated sludge was aerated for one week. The sludge was diluted in the bottles to 2.0 mg/L (van Ginkel and Stroo, 1992). The inoculum was not pre-adapted to the test substance.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Deionized water
Deionized water containing no more than 0.01 mg/L Cu (ISO/IEC 17025; non-GLP analysis) was prepared in a water purification system.

Test bottles
The test was performed in 0.30 L BOD (biological oxygen demand) bottles with glass stoppers.

Nutrients, and stocks
The nutrient medium of the Closed Bottle test contained per litre of deionized water; 8.5 mg KH2PO4, 21.75 mg K2HPO4, 33.4 mg Na2HPO4.2H2O, 22.5 mg MgSO4.7H20, 27.5 mg CaCl2, 0.25 mg FeCl3.6H2O. Ammonium chloride was omitted from the medium to prevent nitrification.
Accurate administration of the water-insoluble test substance was accomplished by dissolving 1.0 g/L of the test substance in dichloromethane (DCM). The test substance in DCM (0.6 mL) was directly added to the bottles. The solvent was allowed to evaporate by placing the bottles on a roller bank in a ventilated hood for 4 hours to obtain an even distribution of the test substance on the walls of the bottles. Next the bottles were filled with nutrient medium with inoculum and closed. The resulting concentration of test substance in the bottles was 2.0 mg/L. Sodium acetate was added to the bottles using a stock solution of 1.0 g/L.

Test procedures
The Closed Bottle test was performed according to the study plan. The study plan was developed from ISO Test Guidelines (1994). Use was made of 10 bottles with test substance and inoculum, 10 bottles treated with DCM (added and evaporated) with inoculum, 10 bottles only containing inoculum and 6 bottles with sodium acetate and inoculum.
The concentrations of the test substance, and sodium acetate in the bottles were 2.0 and 6.7 mg/L, respectively. Each of the prepared solutions was dispensed into the respective group of BOD bottles so that all bottles were completely filled without air bubbles. The zero time bottles were immediately analyzed for dissolved oxygen using an oxygen electrode. The remaining bottles were closed and incubated in the dark. Two duplicate bottles of all series were withdrawn for analyses of the dissolved oxygen concentration at day 7, 14, 21, and 28.

Calculation of the results
Calculation of endogenous respiration
The endogenous respiration (oxygen depletion in the control) was calculated as follows;
Oxygen depletion (endogenous respiration) (mg/L) = Mc (day 0) - Mc (day 28)
MC is the mean oxygen level in the control bottle with mineral salts medium and inoculum.

Calculation of the theoretical oxygen demand (ThOD)
The ThODs of Dichlorobis(n-cyclopentadienyl)titanium, and sodium acetate were calculated
from their molecular formulae and molecular weights as follows.
ThOD_NH3(mgO2 / mg) = (16(2C+0.5(H-Cl-3N)+3S+2.5P+0.5Na-O)/MW

Calculation of the biochemical oxygen demand (BOD)
Provided that the oxygen concentrations in all bottles at the start of the test were equal, the amounts of oxygen consumed in test and reference compound bottles were calculated as follows:
Oxygen consumption (mg/L) by test substance = MCS - Mt
Oxygen consumption (mg/L) by reference compound = Mc- Ma
Mc or cs the mean oxygen level in control bottles (standard) and control bottles treated with DCM (added and evaporated) n-days after the start of the test.
Mt or a is the mean oxygen concentration in the bottles containing the test substance (t) or the reference compound, sodium acetate (a), n-days after the start of the test.
The biological oxygen demand (BOD) mg/mg of the test substance and sodium acetate was
calculated by dividing the oxygen consumption by the concentration of the test substance and
sodium acetate in the closed bottle, respectively.

Calculation of the biodegradation percentages
The biodegradation was calculated as the ratio of the biochemical oxygen demand (BOD) to
the theoretical oxygen demand (ThOD).
Reference substance:
acetic acid, sodium salt
Test performance:
The pH of the media was 7.3 at the start of the test. The pH of the media at day 28 was 7.4 (test) and 7.3 (control (DCM added and evaporated) and standard control). Temperatures were within the prescribed temperature range of 22 to 24°C.
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Details on results:
Theoretical oxygen demand (ThOD)
The calculated theoretical oxygen demand (ThOD) of the test substance is 1.7 g oxygen/g test substance. The ThOD was calculated by assuming that Ti was oxidized to TiO2. The ThOD of sodium acetate is 0.8 mg/mg.

Toxicity
Inhibition of the degradation of a well-degradable compound, e.g. sodium acetate by the test substance in the Closed Bottle test was not determined because possible toxicity of
dichlorobis(η-cyclopentadienyl)titanium to microorganisms degrading acetate is not relevant. Inhibition of the endogenous respiration of the inoculum by the test substance at day 7 was not detected. Therefore, no inhibition of the biodegradation due to the "high" initial test substance concentration is expected.

Biodegradability
Dichlorobis(n-cyc|opentadienyl)titanium was not biodegraded (0% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
Parameter:
ThOD
Value:
1.7 g O2/g test mat.
Results with reference substance:
The validity of the test is demonstrated by an endogenous respiration of 0.8 mg/L at day 28. Furthermore, the differences of the replicate values at day 28 were less than 20%.
The biodegradation percentage of the reference compound, sodium acetate, at day 14 was 80. Finally, the validity of the test is shown by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
The test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Dichlorobis(η- cyclopentadienyl)titanium was not biodegraded (0% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.
The test is valid as shown by an endogenous respiration of 0.8 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 80% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.
Executive summary:

In order to assess the biotic degradation of the test substance, a ready biodegradability test was performed which allows the biodegradability to be measured in an aerobic aqueous medium. The ready biodegradability was determined in the Closed Bottle test performed according to slightly modified OECD, EU and ISO Test Guidelines, and in compliance with the OECD principles of Good Laboratory Practice.

The test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Dichlorobis(η- cyclopentadienyl)titanium was not biodegraded (0% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

The test is valid as shown by an endogenous respiration of 0.8 mg/L and by the complete degradation of the reference compound, sodium acetate. Sodium acetate was degraded by 80% of its theoretical oxygen demand after 14 days. Finally, the most important criterion was met by oxygen concentrations >0.5 mg/L in all bottles during the test period.

Description of key information

The test item did not cause a reduction in the endogenous respiration at day 7. The test substance is therefore considered to be non-inhibitory to the inoculum. Dichlorobis(η- cyclopentadienyl)titanium was not biodegraded (0% after 28 days) in the Closed Bottle test and should therefore not be classified as readily biodegradable. The lack of biodegradation in the Closed Bottle test does not mean that the test substance is recalcitrant in nature because the stringency of the test procedures could account for the recalcitrance in the Closed Bottle test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information